RORα In Macrophages Senses IGF1/AMPK Signaling To Boost Diabetic Bone Regeneration By Recruiting BMSCs

Objective: The aim of this research was to explore the effects and potential molecular mechanisms of retinoic acid receptor-related orphan receptor α(RORα)in macrophages during bone regeneration in type 2 diabetes mellitus(T2DM),which may bring a new strategy for improving the diabetic craniofacial bone regeneration.Methods: After constructing normal and T2 DM rat cranial defect models,transcriptome sequencing(RNA-seq)technology was applied to detect the expression characteristics of enrichment pathways and related rhythm genes in regenerated bone tissues of both groups.In 13-week-old T2 DM rats,SR1078 was injected intraperitoneally to activate the downstream effects of RORα protein,and a cranial bone defect model was constructed 48 hours later.Cranial bone tissues were collected after 7d,14 d and 28 d,and the effects of RORα agonist SR1078 on in situ bone regeneration in T2 DM were examined using microcomputed tomography(Micro-CT),immunohistochemistry(IHC)staining and real-time fluorescence quantitative reverse transcription polymerase chain reaction(q RT-PCR)methods.Immunofluorescence(IF)was used to observe the changes of RORα expression in macrophages during different stages of bone defect repair in diabetic and normal rat models.Normal SD rats were treated with SR3335 to inhibit the expression of RORα and made into the same model of calvarial defects.Correlation detection was aimed to further explore the sensibility of RORα to perceive the inflammatory microenvironment and the influence on osteogenesis.High-glucose microenvironment was constructed in vitro.THP-1,a human monocyte-derived cell line,were treated with IGF1 protein,IGF1 R and related signaling pathway inhibitor.The changed expression levels of RORα protein were detected by western blot(WB)and immunofluorescence(IF)assays.Meanwhile,IGF1 loaded in Gel MA was applied topically in the calvarial defect in rats with T2 DM.The Rorα expression in bone tissue and the formation of new bone can be observed by q-PCR and Micro-CT analysis.We cultured primary bone marrow derived macrophages(BMDMs)and bone marrow mesenchymal stem cells(BMSCs).Through constructing over-expression or silencing vector,Rorα was overexpressed or knocked down in BMDMs.Cellular supernatant of Rorα-overexpressing or Rorα-knockdown BMDMs was used as conditioned medium to induce the migration and proliferation experiments of BMSCs.We screened the downstream target gene,of which transcription may be affected by RORα through retrieving databases and validated by q RT-PCR.The inner relation between RORα and BMSCs was exploited by Chromatin Immunoprecipitation(Ch IP)and functional experiments,including migration and proliferation.Results: 1.Activation of RORα can promote in situ bone regeneration in diabetes;2.In early bone repair,the expression of RORα is high in macrophages.In the normal physiological conditions,the expression of RORα increased significantly at first and then decreasing,while it can not be raised in diabetes;3.The application of inhibitors disturbed the functional upregulation of RORα in early bone repair under normal conditions and the repair capacity of bone defect decreased obviously,indicating that the upregulation of RORα in macrophages could remarkably promote bone regeneration in the early stage;4.Insufficient IGF1-AMPK signaling in diabetic microenvironment impeded the upregulation of RORα;5.RORα in BMDMs actuated the proliferation and migration of BMSCs through upregulation of CCL3 and IL-6.Conclusion: The circadian nuclear receptor RORα,which is a key transcription factor in dominating the cell interaction between macrophages and other related cells,can also influence the extracellular environment.The upregulation of RORα in macrophages is significant for physiological in situ bone regeneration,promoting the transcription of cytokines secreted from macrophages and thereby increasing the recruitment and proliferation of BMSCs.Though applying RORα agonist,the abnormal expression of RORα in diabetes could be altered,providing a new strategy for the tough problem in diabetic bone regeneration.